The Role of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in Haematopoiesis

Yi Shen (2010). The Role of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in Haematopoiesis PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Yi Shen
Thesis Title The Role of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in Haematopoiesis
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2010-05
Thesis type PhD Thesis
Supervisor Associate Professor Jean-Pierre Levesque
Dr Ingrid Winkler
Total pages 254
Total colour pages 45
Total black and white pages 209
Subjects 11 Medical and Health Sciences
Abstract/Summary Haematopoietic stem cells (HSC) give rise to haematopoietic progenitor cells (HPC) which terminally differentiate into all blood cell types. HSC can reconstitute the haematopoietic system following damage and reside in specific micro-domains of the bone marrow (BM) known as niches. HSC niches are preferentially located at the interface between the bone and BM (endosteum) and are abundant in long bones. Molecules expressed in these niches provide the extrinsic cues that control HSC quiescence, self-renewal or differentiation by asymmetric division. Many proteins found in these niches are involved in the egress of HSC into the blood during mobilisation. These proteins include proteases such as neutrophil elastase, cathepsin-G and matrix metalloproteinases (MMP), as well as their inhibitors, α1-antitrypsin (serpina1) and tissue inhibitor of metalloproteinases (TIMP)-1 to -4. TIMP-3 is a less studied member of the TIMP family. It not only regulates MMP activity but also the activity of sheddases (and thus plays a role in cytokine maturation), prevents vascular endothelial growth factor binding to its receptor 2, inhibits cancer proliferation and can cause sorsby fundus dystrophy. Despite its many interesting functions, TIMP-3’s role in haematopoiesis and mobilisation has not been well documented. By qRT-PCR and reverse zymography, I have determined that TIMP-3 mRNA is expressed at a 10-fold higher level at the endosteum when compared to central BM. Multipotent stromal cells (MSC), endothelial cells and mature osteoblasts (OB) expressed high levels of TIMP-3. Erythroid progenitors, megakaryocyte progenitors and mature megakaryocytes expressed TIMP-3 mRNA at much lower levels than stromal cells, while all other haematopoietic cells expressed extremely low to undetectable levels of TIMP-3 mRNA. Immunohistofluorescence on paraffin embedded bone sections with a rabbit anti-human TIMP-3 antibody (which cross-reacts with mouse TIMP-3) validated TIMP-3 protein expression in endothelial cells, OB and megakaryocytes. Interestingly, TIMP-3 mRNA and protein expression decreased with granulocyte-colony stimulating factor induced mobilisation. To investigate TIMP-3 function, human TIMP-3 (huTIMP-3) cDNA was over-expressed in mouse HSC via retroviral transduction using MND-X-IRES-eGFP (MXIE) retroviral vector. In mice transplanted with HSC transduced with MXIE retroviral vector containing huTIMP-3 cDNA, the frequency of B-cells and T-cells over-expressing huTIMP-3 were significantly reduced in blood and BM when compared to mice transplanted with HSC transduced with control empty vector. Conversely, the frequency of huTIMP-3 expressing myeloid cells was significantly higher in the blood, BM and spleen. Blood leukocytes and splenocytes of mice over-expressing huTIMP-3 in haematopoietic cells exhibited significantly greater colony forming ability. The presence of huTIMP-3 also increased Lineage-Sca-1+Kit+ stem cell proliferation both in vivo and in vitro. Interestingly as well, long term over-expression of huTIMP-3 resulted in calcification of bones with markedly decreased BM content and ultimately death.
Keyword bone marrow
haematopoiesis
haematopoietic stem cells
mobilisation
niches
osteoblasts
TIMP-3
Additional Notes Coloured pages: 34 41 47 52 70 77 104-106 110-113 124-126 128-130 134 137 139-141 145-147 149-150 160 165 170-174 176-178 209-204

 
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